Recent clinical trials have shown that antiestrogens can reduce the risk of invasive breast cancer in high-risk women without existing cancer. However, antiestrogens do not reduce the incidence of estrogen receptor negative breast cancers in these women. Thus, there is an urgent need to identify and test agents that will prevent the development of ER-negative breast cancer. While ER-negative breast cells do not respond to estrogen, they do require growth factors such as EGF, TGFalpha, and IGF-I, so that the molecules that transduce these growth factor signals are targets for agents for tile treatment and prevention of ER-negative breast cancer. Particularly promising agents for the prevention of ER-negative breast cancer include retinoids and signal transduction inhibitors, such as tyrosine kinase inhibitors and COX-2 inhibitors. These agents all suppress the growth of cancer cells in vitro, and will inhibit the growth of normal and malignant breast cells. In addition, our preliminary results suggest that these agents can suppress the development of breast cancer in vivo in mouse models that develop ER-negative breast cancer. We now propose three aims to test the hypothesis that successful chemoprevention by these agents will be associated with modulation of specific biomarkers in the mammary gland tissue. We will: 1) compare the ability of molecularly targeted chemo-preventive agents to suppress the development of ER-negative breast cancer in three mouse models relevant to human ER-negative breast cancer (C3(1)-SV40Tag, MMTV-c-erbB2, and p53 null mammary gland transplant models); 2) identify by genomic (SAGE) and proteomic techniques mammary tissue biomarkers that are regulated by chemopreventive agents found to effectively suppress ER-negative mammary tumor formation in the mouse models; and 3) develop RNA- and protein-based assays for these markers and validate the modulation of these biomarkers by effective agents in mouse mammary glands and human breast cells. These biomarkers will then serve as markers in future chemoprevention clinical trials to test promising molecularly targeted agents.